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PubMed2003

Overexpression of the complementary DNA for human glutamine:fructose-6-phosphate amidotransferase in mesangial cells enhances glucose-induced fibronectin synthesis and transcription factor cyclic adenosine monophosphate-responsive element binding phosphorylation.

Hyperglycemia-induced alterations in mesangial cell function and extracellular matrix protein (ECM) accumulation are seen in diabetic glomerulopathy. The hexosamine biosynthesis pathway (HBP) is implicated in mediating several metabolic effects of high glucose (HG) in cells. This pathway converts fructose-6-phosphate to glucosamine (GlcN)-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFA). We have previously shown that metabolism of glucose through the HBP regulates the effects of glucose on ECM (fibronectin) synthesis and transcription factor (cyclic adenosine monophosphate-responsive element binding [CREB]) phosphorylation in SV-40-transformed rat kidney mesangial cells. UDP-N-acetyl-GlcN is the end product of the HBP and serves as a precursor for O-linked serine/threonine glycosylation of cytoplasmic and nuclear proteins. Here we show that culturing mesangial cells in HG and GlcN increases the level of O-N-acetylglucosamine in several cytoplasmic and nuclear proteins. Inhibition of O-glycosylation by benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside blocks both HG and GlcN-induced fibronectin synthesis and CREB phosphorylation. To further support the hypothesis that the HBP mediates HG-induced ECM synthesis, a complementary deoxyribonucleic acid (DNA) for human GFA was stably expressed in mesangial cells. Mesangial and GFA-overexpressing cells were cultured in 5 to 25 mM glucose for 48 hours. GFA-overexpressing cells were more sensitive to glucose as they demonstrated increases in fibronectin and CREB phosphorylation at lower glucose concentrations than seen In control cells. In addition, the response to 25 mM glucose for both proteins was increased in GFA when compared with controls. There is no difference in DNA synthesis and cellular adenosine triphosphate levels between the two cell lines. These results suggest that the HBP is a glucose sensor and mediator of the effects of hyperglycemia in the diabetic mesangium.

Animals↗

PubMed1992

Rapid assembly and disassembly of complementary DNA strands through an equilibrium intermediate state mediated by A1 hnRNP protein.

A1 hnRNP protein, which rapidly renatures complementary strands of nucleic acids in vitro, affects both the equilibrium and kinetic properties of the reaction (single-stranded DNA in equilibrium with double-stranded DNA). A1 lowers the melting transition of duplex DNA. However, at temperatures above this new Tm, both single- and double-stranded DNAs are present at equilibrium and are rapidly interconverting. Although the ratio of single and double strands under these conditions is a function of both the A1 protein and complementary DNA strand concentrations, it is not strongly affected by further increases in temperature. These surprising results demonstrate that A1 does not act as a simple catalyst in promoting renaturation and indicate how A1 and other proteins could act to speed the turnover of intermediate complexes in important biological processes.

DNA↗

PubMed1990

Cloning and characterization of a vertebrate cellular myosin regulatory light chain complementary DNA.

We have isolated two series of complementary DNAs (cDNAs) from a chicken gizzard cDNA library encoding two isoforms of phosphorylatable myosin regulatory light chain (RLC). One of the cDNAs encodes a previously isolated smooth muscle myosin RLC (also referred to as LC20-A); the other encodes a protein that shares 92% homology with the LC20-A isoform. The phosphorylatable threonine and serine residues at positions 18 and 19 of the two myosin RLC sequences are conserved. The two cDNAs are 81% homologous at the nucleotide level over the coding region; the 5' and 3' untranslated regions are divergent. Most of the DNA nonhomology in the coding region does not affect the protein sequence, indicating strong evolutionary conservation pressure to maintain the myosin RLC structure. Northern blot analysis using 3' untranslated region probes reveals restrictive tissue specific expression of one myosin RLC isoform (LC20-A) in smooth muscle tissue and not in other tissues examined. In contrast, the novel myosin RLC isoform messenger RNA (mRNA) is uniformly expressed in all smooth and nonmuscle tissues examined and is designated as cellular myosin RLC for this reason. Our results indicate that cellular and smooth muscle myosin RLC isoforms are distinct and are encoded by separate genes. This report describes the cloning of a novel vertebrate cellular myosin RLC mRNA that differs from previously characterized smooth muscle RLC isoform mRNAs in both primary sequence and expression pattern.

Amino Acid Sequence↗

PubMed1995

The tpa-1 gene of Caenorhabditis elegans encodes two proteins similar to Ca(2+)-independent protein kinase Cs: evidence by complete genomic and complementary DNA sequences of the tpa-1 gene.

The gene tpa-1 on chromosome IV of the nematode Caenorhabditis elegans plays a major and definitive role in the adversary action of tumour-promoting phorbol esters, which induce growth arrest and locomotory distress in the animal. The gene was deduced to code for a protein kinase C (PKC) homologue by molecular cloning. We have now sequenced the complete genomic and complementary DNAs for tpa-1 and have analysed their structural features in detail: (1) tpa-1 spans over 20 kb consisting of eleven exons and ten introns; (2) two different-sized mRNAs are generated from the tpa-1 locus; (3) both mRNAs are trans-spliced to the trans-spliced leader SL1; (4) both mRNAs encode PKC isoforms, which are most similar to Ca(2+)-independent novel PKC0; (5) the two PKC isoforms differ from each other in that the smaller lacks the amino-terminal region of the larger corresponding to the first four exons and a portion of the fifth exon; and (6) three introns are located at; identical positions in the polypeptide sequences aligned between the C. elegans tpa-1 product and a PKC of the fruit fly Drosophila melanogaster.

Amino Acid Sequence↗

PubMed2003

Real-time detection of nucleotide incorporation during complementary DNA strand synthesis.

Real-time observation of DNA strand synthesis by using a supercritical angle fluorescence detection apparatus for surface-selective fluorescence detection is described. DNA template molecules were immobilized on a glass surface and the synthesis of the complementary strand was observed after addition of enzyme, dTTP, dATP, dGTP, and fluorescently labeled dCTP (d, deoxy; TP, triphosphate; T, A, G, and C, nucleobases). The fluorescence increase during the Klenow-fragment-catalyzed polymerization depends on the number of labeled dCTP nucleotides incorporated. The efficiency of this reaction is of the same order of magnitude as that of a bimolecular hybridization reaction.

Base Sequence↗

PubMed1987

[Cloning of DNA complementary to mRNA for proopiomelanocortin from the bovine, rat and human hypophysis. Hormonal regulation of proopiomelanocortin mRNA in the rat hypophysis].

Cloning of DNA and complementary mRNA of bovine, rat and human proopiomelanocortin (POMC) was carried out. A structural analysis of the cloned cDNA of POMC was performed. Using restriction fragments of bovine, rat and human POMC cloned cDNA, probes for molecular hybridization based on one-chain bacteriophage M13 were made. Using the dot-hybridization technique with labeled [32P] POMC cDNA, the effect of 17 beta-estradiol and adrenalectomy on the POMC mRNA level in rat hypophysis was studied. The hormone was shown to significantly decrease the POMC mRNA content at a concentration of 4 and 8 micrograms per 100 g of body weight 4 hours after injection. Adrenalectomy caused a 2-3-fold increase in the POMC mRNA level in rat hypophysis after a period of 5-7 days. The possibility of polyhormonal control of POMC genome transcription is discussed.

Adrenalectomy↗

PubMed2005

Evidence for the regulation of alternative splicing via complementary DNA sequence repeats.

MOTIVATION: While the mechanism for regulating alternative splicing is poorly understood, secondary structure has been shown to be integral to this process. Due to their propensity for forming complementary hairpin loops and their elevated mutation rates, tandem repeated sequences have the potential to influence splicing regulation. RESULTS: An analysis of human intronic sequences reveals a strong correlation between alternative splicing and the prevalence of mono- through hexanucleotide tandem repeats that may engage in complementary pairing in introns that flank alternatively spliced exons. While only 44% of the 18 173 genes in the Human Alternative Splicing Database are known to be alternatively spliced, they contain 84% of the 694 237 intronic complementary repeat pairs. Significantly, the normalized frequency and distribution of repeat sequences, independent of their potential for pairing, are indistinguishable between alternatively spliced and non-alternatively spliced genes. Thus, the increased prevalence of repeats with pairing potential in alternatively spliced genes is not merely a consequence of more repeats or repeat composition bias. These results suggest that complementary repeats may play a role in the regulation of alternative splicing. CONTACT: harold.garner@utsouthwestern.edu.

Alternative Splicing↗

PubMed1993

Nucleosome assembly during complementary DNA strand synthesis in extracts from mammalian cells.

Circular single-stranded phage M13 DNA is used as a template for complementary strand synthesis in cytosolic extracts from proliferating HeLa cells. DNA synthesis is initiated by one or maximally two priming events and typically leads to covalently closed double-stranded reaction products. When carried out in the presence of the nuclear chromatin assembly factor CAF-1, complementary strand synthesis is accompanied by nucleosome assembly. This novel system is very useful for the study of basic biochemical aspects concerning the assembly of nucleosomes. The activity of CAF-1 completely depends on complementary strand synthesis and acts stoichiometrically to promote the assembly of nucleosomes in a noncooperative manner. Apparently, CAF-1 activity is coupled to DNA synthesis via a structural feature of replicating DNA, most likely its partial single strandedness.

Bacteriophage M13↗

PubMed1975

Quantitative and qualitative differences in DNA complementary to avian myeloblastosis virus between normal and leukemic chicken cells.

Hybridization of avian myeloblastosis virus (AMV) RNA with DNA immobilized on filters or in liquid with a vast DNA excess was used to measure the viral specific DNA sequences in chicken cells. Newly synthesized viral DNA (v-DNA) appears within an hour after infection of chicken embryo fibroblasts (CEF) with avian oncornaviruses. A fraction of newly synthesized v-DNA becomes integrated into the cellular genome and the remainder gradually disappears. A covalent linkage between v-DNA and cellular DNA was demonstrated to exist in CEF and in leukemic myeloblasts by alkaline sucrose velocity sedimentation. Hybridization of AMV RNA in DNA excess has revealed that there are 2 clases of viral specific sequences within normal as well as in leukemic cells. The 2 types of sequences differ in their rate of hybridization. The amount of both types of DNA sequences is about 2 times higher in leukemic cells than in normal cells. Both the fast- and slowly reacting sequences in leukemic cells exhibit a higher Tm (2 degrees C) than the respective DNA sequences in normal cells. Furthermore, when nucleotide sequences in AMV RNA complementary to normal DNA are removed first by exhaustive hybridization with normal DNA, the residual RNA only hybridizes with leukemic DNA but not with normal DNA. These results suggest that leukemic cells contain viral specific DNA sequences which are absent in normal cells. Endogenous v-DNA has been shown to be integrated in cellular DNA region(s) with a reiteration frequency of approximately 1,200 copies per cell and each integration unit appears to have a size approximately equivalent to the 35S RNA subunit of the viral genome. Viral sequences acquired after infection appear to be integrated in the unique region of cell DNA, or in tandem with the endogenous viral sequences.

Animals↗

PubMed1993

Renaturation of complementary DNA strands by herpes simplex virus type 1 ICP8.

ICP8, the major single-stranded DNA-binding protein of herpes simplex virus type 1, promotes renaturation of complementary single strands of DNA. This reaction is ATP independent but requires Mg2+. The activity is maximal at pH 7.6 and 80 mM NaCl. The major product of the reaction is double-stranded DNA, and no evidence of large DNA networks is seen. The reaction occurs at subsaturating concentrations of ICP8 but reaches maximal levels with saturating concentrations of ICP8. Finally, the renaturation reaction is second order with respect to DNA concentration. The ability of ICP8 to promote the renaturation of complementary single strands suggests a role for ICP8 in the high level of recombination seen in cells infected with herpes simplex virus type 1.

Animals↗

PubMed1996

Nucleotide sequences of three H-2K and three H-2D complementary DNA clones coding mouse class I MHC heavy chain proteins.

OBJECTIVES: The polymerase chain reaction (PCR)-based method was used to obtain and sequence three H-2K and three H-2D mouse complementary DNAs (cDNA) of class I major histocompatibility complex (MHC) molecules. METHODS: Messenger RNA was isolated from Conconavalin A-activated splenocytes of C57BL/10 (H-2b), C3H (H-2k), and Balb/c (H-2d) mice. We designed H-2K- and H-2D-specific primers as well as a common downstream primer based on previously published mouse class I MHC sequences. Using the PCR method and selective primers we isolated and sequenced H-2Kb and H-2Db cDNAs of C57BL/10, H-2Kk and H-2k cDNAs of C3H, as well as H-2Kd and H-2Dd cDNAs of Balb/c strains. RESULTS: Analysis of the nucleotide sequences documented similarity between our three H-2K cDNA sequences and all mouse MHC class I sequences available in the GenBank. Similarly, our three H-2D sequences were homologous with all mouse class I MHC sequences deposited in the GenBank. Our H-2K and H-2D sequences were also identical to numerous published sequences. CONCLUSIONS: Using these mouse cDNAs, we plan to determine the localization of polymorphic in vivo immunogenic amino acids in class I MHC H-2K and H-2D alloantigens.

Amino Acid Sequence↗

PubMed2005

Rapid amplification of 5' complementary DNA ends (5' RACE).

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs 1-3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target-in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.

Binding Sites↗

PubMed1983

Identification of multiple species of calmodulin messenger RNA using a full length complementary DNA.

Poly(A) RNA from eel electroplax was used to construct a full length cDNA complementary to calmodulin (CaM) mRNA which was cloned in the PstI site of pBR322 DNA. Recombinant plasmids containing sequences complementary to CaM mRNA were identified by hybridization using a 32P-labeled CaM cDNA (pCM109). Nucleotide sequence analysis reveals that the clone from the plasmid pCM116 contains a 5' nontranslated region of 26 nucleotides, the entire coding region, and a 3' nontranslated region of 408 nucleotides. The amino acid sequence deduced from the nucleotide sequence is similar to those previously reported for CaM from other species. Comparison between the nucleotide sequence of the functional domains of the protein shows extensive homology between all four domains. pCM116 was utilized to determine and compare the populations of RNA present in different tissues. In eel electroplax, three species of cytoplasmic RNAs at 820, 1100, and 2000 nucleotides hybridize to the cDNA probe. The nucleus contains an additional CaM RNA molecule of 5500 nucleotides which may represent a primary transcript of the calmodulin gene. Sequence analysis of the 3' noncoding region of pCM116 reveals 3 possible polyadenylation sites (AATAAA) at positions 573, 580, and 855. The mRNA of 820 nucleotides was derived by polyadenylation at the first site whereas the mRNA of 1100 nucleotides was derived by poly(A) addition at position 855. These data are compatible with the idea that at least 2 of the 3 CaM mRNAs in eel electroplax tissue are derived from a single nuclear transcript by differential polyadenylation during processing.

Amino Acid Sequence↗

PubMed1999

Refined high-field NMR solution structure of a binary-addressed pyrene/perfluoro-azide complementary DNA oligonucleotide system shows extensive distortion in the central nick region.

The structural analysis of the photoactivated binary system of complementary-addressing nucleic acid sequences (1:2:3) by high-resolution NMR spectroscopy and restrained molecular dynamics is reported. The binary system comprised a 12 base-pair target DNA sequence, pdGTATCAGTTTCT (1), and two hexanucleotides, (dAGAAACp-L-Az (2) and Pyr-pdTGATAC (3)), complementary to neighbouring sites in the target DNA. Oligonucleotide (2) is conjugated with a p-azidotetrafludrobenzyl group (Az) via a linker group (L), and the other oligonucleotide (3) is equipped with the photosensitizing pyrenyl-1-methylamino group (Pyr). We now extend the structural analysis of 1:2:3, which was previously based on qualitative 2D 1H-NMR data and thermodynamic analysis of complex formation from UV-visible thermal denaturation experiments. In the current work structural refinement was performed by separate molecular dynamics runs for six different starting structures based on 318 proton-proton distance-range constraints, evaluated from the 1H-NOESY spectrum (tau(mix) = 200 ms, 600 MHz) using complete relaxation matrix analysis (NMR/TRIAD/MARDIGRAS). Additional Watson-Crick hydrogen bond restraints were included in the calculations based on the detected signals from the exchangeable protons, using REFOPT(NY) methods. The final averaged structure obtained from the six refined co-ordinate sets showed a considerable degree of axis bend (62.5 degrees) with the bending point in the middle of the duplex in the region of the backbone nick between the two short oligonucleotides. The complex behaves dynamically as the equivalent of two short B-DNA-like duplexes displaying a hinge-like flexing at their junction. In all final structures the Pyr function location was very restricted, the aromatic group lying in the duplex minor groove near residues 4T, 5C and 2T. In contrast, the location of the perfluoroazido group was different in all the final structures, indicating the high flexibility of this group in the duplex. The only feature common to all six final azido group orientations was the outside location on the side of the major groove. The distance between the Pyr and Az groups varied from 11 A to 24 A for the six final structures (17 A, final average structure). The dynamics of duplex denaturation for 1:2:3 was probed by monitoring the temperature-induced NMR line broadening of the imino protons in a 1D variable temperature NMR experiment. The melting of 1:2:3 starts both from the ends and from the middle part of the duplex at the backbone break between the two short oligonucleotides reflecting the destabilisation of the pyrene-arylazido nick region in the duplex.

Azides↗

PubMed2005

Gene expression profiles in varicose veins using complementary DNA microarray.

BACKGROUND: There has been little information reported about the genetic event concerning the pathophysiology of varicose vein (VV). OBJECTIVES: The purpose of this study was to examine the differentiation of gene expression in the wall of VV using complementary deoxyribonucleic acid (cDNA) microarrays. METHODS: The study was performed with four pairs of VVs and control veins (CVs). cDNA specimens of VVs were prepared from the ribonucleic acid-isolated VVs of patients who underwent venous obliteration, using radiofrequency, as well as from CVs of those who underwent aortocoronary bypass grafting. Each set of VVs and CVs was hybridized with high-density microarray containing 3,063 human cDNAs. The finding of microarray hybridization were scanned, analyzed, and classified with the cluster program. RESULTS: Among 3,063 cDNA clones, 82 genes were up-regulated in VVs, and some of the up-regulated genes, which were detected by cDNA microarray, including transforming growth factor 3-induced gene (BIGH3), tubulin, lumican, actinin, collagen type I, versican, actin, and tropomyosin, belonged to extracelluar matrix molecules, cytoskeletal proteins, or myofibroblasts. CONCLUSIONS: Many up-regulated genes were found in Ws by applying cDNA microarray. These gene profiles suggested a pathway associated with fibrosis and that wound healing might be related to the pathophysiology of VVs.

DNA, Complementary↗

PubMed1997

Carbonic anhydrase IX, MN/CA IX: analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts.

BACKGROUND & AIMS: CA IX (formerly MN protein) is a carbonic anhydrase isoenzyme whose expression is associated with human tumors. However, it has also been found in normal gastric mucosa. The aim of this study was to determine differences in complementary DNAs (cDNAs), to obtain an overview of distribution in the alimentary tract, and to obtain data on expression in tumors. METHODS: A CA9 cDNA isolated from a human stomach library was sequenced along with the cDNA derived from HeLa cells. Western blotting and immunohistochemical analyses of human and animal tissues were performed using CA IX-specific monoclonal antibody and rabbit antiserum to human CA II. RESULTS; Sequence analysis showed no differences between the stomach- and HeLa-derived cDNAs. CA IX was detected at the basolateral surface of gastric, intestinal, and gallbladder epithelia. In stomach tumor samples, expression of CA IX was lost or reduced. CONCLUSIONS: Differential distribution of CA IX in normal and tumor tissues is not associated with cDNA mutations. Evolutionary conservation in vertebrates as well as abundant expression of CA IX protein in normal human gastric mucosa, but not in derived tumors, indicate its physiological importance.

Animals↗

PubMed1984

Use of complementary DNA oligomers to probe trp leader transcript secondary structures involved in transcription pausing and termination.

DNA oligomers were synthesized that are perfectly complementary to different segments of the tryptophan (trp) operon leader transcript. These 15 nucleotide long oligomers were used as probes of the involvement of transcript secondary structures in two processes: transcription pausing at the pause site located near base pair 90 in the leader region, and transcription termination at the attenuator. The 15-mers were complementary to the four segments of the trp leader transcript which have been shown to form the alternative secondary structures that are believed to be responsible for pausing, termination, and antitermination. Oligomers complementary to RNA segments 1 and 3 relieved termination while the 15-mer complementary to RNA segment 1 relieved pausing. 15-mers complementary to segment 2 had no effect on pausing and the oligomer complementary to segment 4 had virtually no effect on termination.

Base Sequence↗

PubMed1983

Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein.

A complementary (cDNA) molecule encoding the structural proteins of Semliki Forest virus (SFV) has been inserted into a Simian virus 40-derived eucaryotic expression vector lacking introns. Introduction of the recombinant DNA into nuclei of baby hamster kidney cells results in the synthesis of authentic SFV membrane glycoproteins E1 and E2. The glycoproteins are both transported to the cell surface and induce cell-cell fusion after a brief treatment of the cells with low pH medium. The pH dependence of the fusion reaction was the same as that induced by virus particles (White, J., J. Kartenbeck, and A. Helenius, 1980, J. Cell Biol., 89:674-679). Transfection of cells with another recombinant DNA molecule in which the SFV cDNA is engineered into the same expression vector including an intron has been shown before to result in the expression of only the E2 protein on the cell surface, whereas the E1 protein is trapped in the rough endoplasmic reticulum (Kondor-Koch, C., H. Riedel, K. Söderberg, and H. Garoff, 1982, Proc. Natl. Acad. Sci. USA, 79:4525-4529). Such cells do not exhibit pH-dependent polykaryon formation, suggesting that the E1 protein is necessary for fusion activity. Immunoblotting experiments show that the RER-trapped E1 protein expressed from the DNA construction with an intron has a smaller apparent molecular weight than authentic E1, and that is has lost its amphipathic characteristics.

Animals↗